Background[unreadable] [unreadable] Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by self-limited attacks of fever with serosal, synovial, or cutaneous inflammation, sometimes complicated by systemic amyloidosis. In 1992 our laboratory mapped the FMF locus to chromosome 16p13.3, and in 1997 we isolated the underlying gene, MEFV, and demonstrated that it is highly expressed in granulocytes. During the 9 years leading up to the present reporting period, we have studied FMF population genetics, the regulation of FMF gene expression in leukocyte subpopulations, the biochemistry and cell biology of pyrin (the FMF protein), and the development of animal models of FMF. The N-terminal 90 amino acids of pyrin comprise a motif, commonly called the PYRIN domain, that is found in approximately 20 human proteins. We and others have demonstrated the cognate interaction of the PYRIN domain of pyrin to the homologous domain of the apoptosis-associated specklike protein with a CARD (ASC), placing pyrin upstream in the regulation caspase-1-mediated interleukin-1 (IL-1) beta activation. In studies of peritoneal macrophages from a mouse strain expressing a truncated form of pyrin, we found increased caspase-1 activation and IL-1 beta processing, and impaired apoptosis through a caspase-8-dependent, IL-1 beta-independent pathway. By yeast two-hybrid studies, we demonstrated that pyrin interacts with the cytoskeletal protein PSTPIP1, and that PSTPIP1 mutations associated with the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA) lead to markedly increased pyrin-binding and IL-1 beta activation. During the previous reporting period we found that the B30.2 domain of pyrin directly interacts with caspase-1 and thereby regulates IL-1 beta activation.[unreadable] [unreadable] Results of the Last Year[unreadable] [unreadable] Cleavage of pyrin by caspase-1: Following up on the previous years observation that pyrin interacts directly with caspase-1, we hypothesized that pyrin might itself be a caspase-1 substrate. We found that pyrin is cleaved by caspase-1 at Asp330, a site remote from the B30.2 domain. Pyrin variants harboring FMF-associated B30.2 mutations were cleaved more efficiently than wild type (WT) pyrin, possibly due to the diminished ability of these mutants to bind and inhibit caspase-1. The N-terminal cleaved fragment interacted with the p65 subunit of NF kappa B and with I kappa B alpha through its 15 aa bZIP basic domain and C-terminal adjacent sequences, respectively, and translocates to the nucleus. The interaction of the N-terminal fragment with p65 enhanced the entrance of p65 into the nucleus, and the interaction with I kappa B alpha induced the calpain-mediated degradation of I kappa B alpha, both of which activate NF kappa B targets. Moreover, absolute and relative quantities of cleaved pyrin and I kappa B alpha degradation products were substantially increased in leukocytes from FMF patients compared with healthy controls. These data support the likelihood of a new pyrin/caspase-1 pathway for NF-kappa B activation.[unreadable] [unreadable] Studies of murine models of FMF: During the last year we have continued characterization of pyrin null mice. Just as the peritoneal macrophages from pyrin-truncation mice are hyperresponsive to bacterial LPS, peritoneal macrophages stimulated with TLR 1, 2, 4, 6, and 7 ligands exhibited increased caspase-1 activation and IL-1 beta production, relative to WT. Studies of the knockout mice also suggest a role for pyrin in the regulation of the cytoskeleton. We found that actin coimmunoprecipitated with pyrin in WT macrophages. In cells from pyrin null mice, there was an increased interaction of actin with filamin, an intracellular actin-binding protein, and with certain bacterial proteins, such as Staphylococcal protein A and Streptococcal protein G. Since the recognition of the cytoskeleton by bacterial protein is an important mechanism for bacterial invasion into cells, these data suggest a role for pyrin as a defense molecule that protects the cytoskeleton from recognition by bacterial proteins. We have also generated knockin mice for the M680I, M694V, and V726A mutations, and have now achieved germline transmission for mice harboring the WT human B30.2 domain. We have observed marked inflammatory phenotypes in all 3 knockin strains, but the interpretation awaits the characterization of the WT knockin.[unreadable] [unreadable] RNA interference of MEFV in human monocytic cells: In order to explore the physiologic role of pyrin human leukocytes, we studied gene expression profiles in human THP.1 monocytic cell lines with and without siRNA-mediatiated inhibition of MEFV. Using Affymetrix cDNA microarray analysis, we identified genes that were differentially expressed in siMEFV-treated cells relative to the scramble-control (SC). Among the genes downregulated in pyrin knockdown cells were CD36, LY96, S100A8, CCR1, CD53, TIRAP, DEDD, SGK, and AKT3, while EMP2 and BCL2 were upregulated. Western analysis confirmed the reduction in CD36 protein, an accessory molecule for lipoteichoic acid (LTA) recognition. Moreover, in siMEFV-treated cells, TNF production was decreased upon LTA stimulation. In contrast with the above-noted peritoneal cells from pyrin knockout mice, pyrin knockdown human monocytes exhibited diminished responses to TLR 1, 2, 4, and 6 agonists. BCL2 gene expression and protein were increased in pyrin knockdown THP.1 cells, and, as might be expected, staurosporine-induced apoptosis was inhibited. Promoter analysis revealed that several genes differentially expressed in pyrin knockdown cells share an IRF2 transcription factor-binding site, and differential IRF2 expression was confirmed by microarray and quantitative RT-PCR. These data suggest that, directly or indirectly, endogenous pyrin facilitates signaling and apoptosis in part by its effect on IRF2. [unreadable] [unreadable] Mutational analysis in patients: Substantial numbers of patients with FMF have only a single demonstrable MEFV mutation. We studied 10 such patients, 8 of whom were heterozygous for the M694V mutation. To identify additional mutations, we performed standard capillary-electrophoresis genomic sequencing of the entire coding region, and the entire 15 kb genomic region encompassing MEFV was sequenced using a chip-based resequencing system. A second mutation was not detected in any of the 10 patients, although there were numerous heterozygous SNPs, thus arguing against the possibility of a deletion. These data support the possibility that, under some circumstances, a single MEFV mutation may be sufficient to cause FMF.[unreadable] [unreadable] Development of animal models for PAPA syndrome: During the current reporting period we have developed PSTPIP1 knockout mice, and knockins for the PAPA-associated E250Q mutation. Phenotypic analyses are currently under way.[unreadable] [unreadable] Conclusions and Significance: Biochemical and functional data obtained during the current reporting period corroborate earlier findings implicating pyrin as an important regulator of inflammation. During the next year we plan to continue studies of pyrin biochemistry and function, continue investigations of murine models of FMF, and continue studies of patients with FMF and related phenotypes.